KR101378240B1 - Tuberculosis prevention composition - Google Patents

Abstract

The present invention provides a nucleic acid sequence encoding a secretion signal sequence, a nucleic acid sequence encoding Flt3L (fms-like tyrosine kinase 3 ligand), and an active site mutant of Mycobacterium tuberculosis Mtb32A or Mtb32A. It relates to a nucleic acid having a nucleic acid sequence encoding. In addition, it provides a pharmaceutical composition for preventing tuberculosis comprising the nucleic acid, and more specifically provides a vaccine composition for preventing tuberculosis.

tuberculosis, Mycobacterium tuberculosis, Mtb32A, mutant, mutant, DNA vaccine

Description

A pharmaceutical composition for preventing tuberculosis

The present invention provides a nucleic acid sequence encoding a secretion signal sequence, a nucleic acid sequence encoding Flt3L (fms-like tyrosine kinase 3 ligand), and an active site mutant of Mycobacterium tuberculosis Mtb32A or Mtb32A. It relates to a nucleic acid having a nucleic acid sequence encoding. In addition, it provides a pharmaceutical composition for preventing tuberculosis comprising the nucleic acid, and more specifically provides a vaccine composition for preventing tuberculosis.

One third of the world's population (about 1.9 billion people) is Mycobacterium tuberculosis ), and about 8 million to 10 million new cases occur every year, and about 3 million people die of tuberculosis every year. In recent years, the incidence of tuberculosis has been high in AIDS patients, and AIDS is known as a new source of tuberculosis. In developed countries, tuberculosis patients are spreading due to the spread of HIV infection. The mortality rate from tuberculosis has increased again since the 1990s, mainly due to increased infections caused by multidrug-resistant tuberculosis bacteria and increased tuberculosis frequency due to the spread of HIV infection. There are currently about 50 million patients with multi-drug resistant tuberculosis (MDR-TB) worldwide, but the treatment efficiency is only 50%. In addition, there are currently about 6 million HIV / TB simultaneous infections worldwide, and most of them are estimated to die from tuberculosis.

Mycobacterium tuberculosis is a rod-shaped bacillus with a very slow growth rate. It is an acid-resistant bacterium that can withstand dry conditions, strong acids or alkalis. The path of infection of tuberculosis is that bacteria mixed in the sputum drops of tuberculosis patients float in the air and inhale when they breathe into the lungs. Most of them heal naturally, but 5-10% of people develop tuberculosis and develop when their body's immunity decreases due to complications such as overwork, undernutrition and diabetes (Kochi et al. al ., Lancet 1997, 350: 142; Toossi and Ellner, Pathogenesis of tuberculosis. In: Friedman LN (ed). Tuberculosis: current concepts and treatment. CRC Press: New York, 2001, pp. 19-47). Therefore, it is very important to develop prophylactic and therapeutic vaccines to protect people who are not infected with TB or who are infected with TB.

Bacillus Calmette-Gurin (BCG) is an attenuated live vaccine that reduces the virulence of a bacterium called Mycobacterium bovis and is the most widely used worldwide and is effective in protecting children from widespread tuberculosis. However, its duration is limited to childhood, and it is known that there is little therapeutic effect in adults. Therefore, there is a need for the development of a vaccine that can overcome the shortcomings of BCG and prevent tuberculosis in the long term. In the present invention, BCG was used as one of the positive control groups.

To date, DNA vaccines have been tried as new prophylactic tuberculosis vaccines, and their efficacy has been demonstrated in animal models. Many TB antigens have also been discovered using DNA vaccine techniques. DNA vaccination has proven to be very effective in establishing cellular immune responses, including cytotoxic T lymphocyte (CTL) and Th1 responses, known to be essential for obtaining effective anti-mycobacterial immunity in mice and humans (Bonato VL et al ., Infect Immun , 1998, 66: 169-175). Due to the importance of selecting an appropriate antigen in the introduction of such an efficient immunization technique, antigen discovery has also been actively progressed. For example, prophylactic DNA vaccines expressing antigens 85A, 85B, 65 kDa heat shock protein or PstS-3 have been found to be effective in limiting the growth of M. tuberculosis in mice (Denis O et al . Infect Immun 66: 1527-1533, 1998; Huygen K et al . Nat Med 1996; 2: 893-898; Lozes E et al . Vaccine 15: 830-833, 1997; Kamath AT et al . Infect Immun 67: 1702-1707, 1999; Tascon RE et al . Nat Med 2: 888-892, 1996; Lowrie DB et al . Vaccine 15: 834-838, 1997; Tanghe A et al . J Immunol 162: 1113-1119, 1999). Currently, Ag85A is most reliably recognized as a single antigen in many animal and clinical models (Philip O et al , Nature review microbiology. 2: 930-932, 2004). Therefore, in the present invention, Ag85A was used as a positive control for a single antigen.

On the other hand, recently, there is a case in which an antigen that is not available as a single antigen or does not show efficacy is fused with other antigens. For example, Ag85B-ESAT6 fusion antigen is well known (Delphine Cendron et. al , Eur . J. Immunol . 37. 2007: 549565). Mtb32A is a serine protease, and Mtb32A has been used in combination with other antigens until now because it is difficult to separate and purify it into proteins due to its intrinsic properties (Yasir AW Skeiky et. al , INFECTION AND IMMUNITY , 67: 3998-4007, 1999), for example the M72F (N-terminus of Mtb32A antigen-C-terminus of Mtb32A antigen-C terminus of Mtb32A antigen) fusion antigens are well known (Yasir AW Skeiky et. al , JI 172; 7618-7628, 2004).

However, these fusion antigens were selected as the next best option either by supplementing the ineffectiveness of a single antigen (Ag85B-ESAT6), or by using it in place of its use as a single antigen (Mtb32A of M72F). In addition, the use of such limited antigens is not economical because it always requires verification of the secondary antigens, and immunological and pathological studies by the single antigens themselves are also inefficient.

Under these circumstances, the present inventors have made efforts to develop a vaccine composition for preventing tuberculosis, and as a result, have demonstrated for the first time the induction and defense of tuberculosis immunogenicity by Mtb32A single antigen using genetic improvement technology. In addition, the present inventors found that mutants that neutralize the activity by substituting three amino acids required for Mtb32A enzyme activation are stable even with a single antigen, induce strong immunogenicity, and also have a preventive effect in Mycobacterium tuberculosis animal model. Proved to be excellent.

Furthermore, the present inventors have introduced an antigen engineering technique that can increase the immune response for the formation of a strong and sustained immune response of the antigen, so that the Flt3L (fms-like tyrosine kinase 3 ligand), which is a costimulatory molecule between immune cells, can be obtained. Immune compositions for optimal tuberculosis prevention were prepared by fusion to Mtb32A active site mutants. Experiments were carried out in a mouse model, which resulted in an increase in immune responses such as Mtb32A-specific antibody responses and CTL cell proliferation and prevention and treatment of tuberculosis. The activity was confirmed to be unpredictably high and the present invention was completed.

One object of the present invention, the nucleic acid sequence encoding the secretion signal sequence, and Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) To provide a nucleic acid having a nucleic acid sequence encoding the active region mutants of Mtb32A or Mtb32A.

Another object of the present invention is to provide a nucleic acid sequence encoding a secretory signal sequence, a nucleic acid sequence encoding Flt3L (fms-like tyrosine kinase 3 ligand), and the activity of Mycobacterium tuberculosis Mtb32A or Mtb32A. It is to provide a nucleic acid having a nucleic acid sequence encoding a site mutant.

Still another object of the present invention is to provide a pharmaceutical composition for preventing tuberculosis comprising the nucleic acid.

When a nucleic acid sequence encoding Mycobacterium tuberculosis Mtb32A acting as an antigen is used as a DNA vaccine or the like, the present inventors used a nucleic acid having the following characteristics to enhance its efficacy.

That is, as one embodiment, the present invention provides a Mycobacterium tuberculosis (1) nucleic acid sequence encoding the secretion signal sequence and (2) a nucleic acid sequence linked to the 3 'end of the secretion signal sequence, Mycobacterium tuberculosis ) relates to a nucleic acid having a nucleic acid sequence encoding a mutation of an active site of Mtb32A or Mtb32A.

Preferably, the present invention provides a nucleic acid sequence encoding (1) a secretion signal sequence, (2) a nucleic acid sequence linked to the 3 'end of the secretion signal sequence, Flt3L (fms-like tyrosine kinase 3 ligand) A nucleic acid sequence coding for), and (3) a nucleic acid sequence linked to the 3 'end of the nucleic acid sequence encoding Flt3L, encoding an active site mutant of Mycobacterium tuberculosis Mtb32A or Mtb32A. It relates to a nucleic acid having a nucleic acid sequence.

As used herein, the term "Mtb32A" refers to a 32 kD serine protease present in Mycobacterium tuberculosis. Nucleic acid sequences and amino acid sequences of Mtb32A are known in public genetic databases such as Sanger ID: Rv0125, and are described in prior art (eg US Patent Application No. 60 / 158,585; Skeiky et al., Infection and Immun. 67: 3998-4007, 1999).

As used herein, the term "active site mutant of Mtb32A" is a variant that inhibits the possibility of causing intracellular toxicity by mutating the active site of Mtb32A as a serine protease. Specifically, in the present invention, the 64th amino acid (Histidine) of Mtb32A as alanine, the 95th amino acid (Aspartate) as asparagine (Asparagine), the 177th amino acid (Serine) as alanine (Alanine) as a codon Substituted mutants were used to demonstrate that these mutants reduced the likelihood of causing intracellular toxicity, enhanced intracellular expression rates and induced high immune responses, and induced high tuberculosis prevention effects (FIGS. 1-5).

In the present invention, the active site mutant of Mtb32A or Mtb32A is used as the antigenic protein of Mycobacterium tuberculosis. As used herein, the term “antigen” refers to a molecule capable of generating an immune response, and stimulates the immune response by administering the antigen to an animal including a human.

As used herein, the term "secretory signal sequence" is a molecule that induces the secretion of Mtb32A or active site mutants of Mtb32A out of the cell, the nucleic acid sequence encoding the secretion signal sequence is not particularly limited. In a specific embodiment of the present invention, tPa (tissue plasminogen activator) was used. The nucleic acid sequence encoding the secretion signal sequence is located upstream of the nucleic acid sequence encoding the Mtb32A or Mtb32A active site mutant in which the gene codon is substituted.

In the present invention, the term "fms-like tyrosine kinase 3 ligand" (Flt3L) is a costimulatory molecule between immune cells, and serves to increase the number of dendritic cells involved in the immune response. In the present invention, in order to enhance the tuberculosis prevention effect of the active site mutant of Mtb32A or Mtb32A Flt3L was used in fusion. The nucleic acid sequence encoding the Flt3L is located upstream of the nucleic acid sequence encoding the Mtb32A or Mtb32A active site variant.

In addition, the secretion signal sequence constituting the nucleic acid of the present invention, the nucleic acid sequence encoding Flt3L, and the nucleic acid sequence encoding the active site mutant of Mtb32A or Mtb32A are genes with high expression frequency in mammals, for example, human cells. Substitution with codons can maximize intracellular expression efficiency, referred to as "codon optimization".

One amino acid is encoded by one or more gene codons and translated by tRNA that specifically recognizes it. However, even though the gene codons encoding the same amino acids, the frequency of use of tRNAs for recognizing them in the cell is significantly different. For example, in the case of Ala, they are encoded by GCC, GCT, GCA and GCG, but the probability that these codons are recognized by tRNA in human cells differs significantly. Specifically, the probability that the GCC codon will be recognized by the tRNA that recognizes it is about 53%, but the probability that the GCT (17%), GCA (13%), and GCG (17%) codons will be recognized by the tRNA that recognizes it is Very low Therefore, when the gene codon of the nucleic acid sequence constituting the nucleic acid of the present invention is replaced with a gene codon recognized by tRNA in human cells, the expression level of the nucleic acid in the cell may be significantly increased.

Accordingly, the present invention preferably comprises (1) a nucleic acid sequence encoding a codon optimized secretion signal sequence, (2) a nucleic acid sequence encoding a codon optimized Flt3L, and (3) a codon optimized mycobacterium tuberculosis A nucleic acid having a nucleic acid sequence encoding an active site mutant of Mtb32A or Mtb32A is included.

Specifically, the nucleic acid sequence encoding the secretion signal sequence in the present invention can be used codon optimized, preferably has a nucleic acid sequence of SEQ ID NO: 1. In addition, the nucleic acid sequence encoding the Mtb32A active site mutant is preferably used by codon optimization as shown in SEQ ID NO: 2. In addition, the nucleic acid sequence encoding Flt3L is preferably used by codon optimization as shown in SEQ ID NO: 3.

In the present invention, the fragment can be used as an antigen within the range having the activity as an antigen as well as the entire amino acid sequence of the codon-optimized Mtb32A or Mtb32A active site mutant.

In addition, the nucleic acid can be prepared by artificially synthetic or genetic recombination methods, it is provided by operatively linked to a vector capable of expressing it.

In another aspect, the present invention relates to a pharmaceutical composition for preventing tuberculosis comprising the nucleic acid. As used herein, the term "prevention" refers to any action that inhibits or delays the development of tuberculosis by administration of a composition comprising the nucleic acid.

Preferably, in the present invention, the pharmaceutical composition for preventing tuberculosis containing the nucleic acid may be provided in the form of an expression vector.

As used herein, an "expression vector" refers to a gene construct that is capable of expressing a protein of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express a gene insert. As used herein, the term “operably linked” refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. A promoter and a nucleic acid sequence encoding a protein can be operably linked to affect the expression of the encoding nucleic acid sequence.Operative linkages with recombinant vectors can be prepared using genetic recombination techniques well known in the art. Site-specific DNA cleavage and ligation uses enzymes generally known in the art, and the like.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of the nucleic acid sequence encoding the immunogenic target protein and must be functional in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, includes a replication origin, which is a specific nucleic acid sequence from which replication is initiated. Vectors that can be integrated into the genome itself are also available.

The compositions of the present invention are formulated into suitable formulations with acceptable pharmaceutical carriers and administered by a variety of routes. The route of administration may be oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, topical, nasal, pulmonary, rectal. Preferably, it is formulated as an injection and administered by the method of intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and the like. Injectables include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Preservatives such as mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like. In a specific embodiment of the present invention, it was administered muscle by injection.

The composition of the present invention is administered in an immunologically effective amount. The term "immunologically effective amount" means an amount sufficient to exhibit tuberculosis prevention effect and an amount not to cause side effects or serious or excessive immune response, the exact dosage depends on the specific immunogen to be administered, and Age, weight, health, sex of the subject can be easily determined by those skilled in the art according to factors well known in the medical arts, such as drug sensitivity, route of administration, method of administration, and can be administered from one to several times.

The composition of the present invention may preferably be provided in the form of a vaccine composition. The prophylactic vaccine composition of the present invention can be administered alone but is preferably administered with an adjuvant.

Adjuvant is a substance that promotes an immune response to an antigen in a specific way during the initial activation of immune cells, and means an agent, a molecule, etc. that enhances immunity by increasing the activity of cells of the immune system, although it is not an immunogen to the host (Warren et. al., 1986, Annu. Rev. Immunol ,. 4: 369). Adjuvants that can be administered with the compositions of the present invention to enhance an immune response include any of a variety of adjuvants, and typical adjuvant includes Freund adjuvant, aluminum compound, mura Wheat dipeptide, lipopolysaccharide (LPS), monophosphoryl lipid A, coil A, etc. In the present invention, monophosphoryl lipid A was used as an adjuvant. It may be administered simultaneously with the vaccine prophylactic composition or sequentially at intervals of time.

The composition of the present invention showed a very good effect as a prophylactic vaccine while showing an excellent effect on the production of INF-γ and a reduction in the number of Mycobacterium tuberculosis. Specifically, in the present invention, a composition comprising an expression vector operably linked with a tPa secretion signal sequence, a nucleic acid sequence encoding Flt3L, and a nucleic acid sequence encoding an Mtb32A mutant substituted with the 64th, 95th, and 177th amino acids of Mtb32A. 12 weeks after immunization of mice, the lungs and spleens of the rats were taken out to examine antigen-specific immune responses. As a result, the amount of INF-γ produced was higher than that of the fusion peptides of Ag85A and Flt3L, which are known TB antigens. It was proved effective in inducing an immune response (FIG. 3). In addition, after immunizing the mice with the composition and infecting Mycobacterium tuberculosis 4 and 16 weeks later, the number of Mycobacterium tuberculosis in the lungs and spleen of the rats was compared. As a result, the most distinct bacterial count was compared with the fusion peptide of Ag85A and Flt3L or BCG vaccine. Showed a decrease. Tuberculosis prophylaxis vaccines require long-term continuous prevention rather than short-term prevention, so the composition of the present invention can be usefully used for the prevention of tuberculosis (FIGS. 4 and 5).

As described above, by administering a nucleic acid comprising a nucleic acid sequence encoding a secretion signal sequence, a nucleic acid sequence encoding Flt3L, and a nucleic acid sequence encoding a Mtb32A or Mtb32A active site variant, Mycobacterium It can effectively prevent and treat tuberculosis, a disease caused by tuberculosis infection.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example 1: Construction of pGX10 / tMtb32Amut DNA

The codon optimized tPa secretion signal sequence having the nucleic acid sequence of SEQ ID NO: 1 was chemically synthesized. KpnI (5 ') and Eco47III-NheI (3') sites were added at the ends. Codon-optimized Mtb32Amut having the nucleic acid sequence of SEQ ID NO: 2 was chemically synthesized, and NheI-EcoRV (5 ') and SpeI-AscI-XbaI (3') sites were added at the ends to facilitate insertion into the vector. In order to prevent the possibility of Mtb32A's intracellular toxicity, the 64th amino acid (Histidine) of Mtb32A is alanine, the 95th amino acid (Aspartate) is asparagine, and the 177th amino acid (Serine) is alanine ) To command codon. N-termina HA (hemagglutinin) tag having the nucleic acid sequence of SEQ ID NO: 4 was chemically synthesized. AscI (5 ') and NotI-BstXI-XbaI (3') sites were added at the ends. To facilitate expression of Mtb32A, the codon was repeated twice at the terminal of the Mtb32A antigen.

PGX10 (Patent No. 2003-0047667), a DNA vaccine preparation vector, was treated with KpnI and NheI enzymes (enzyme), and then tPa, a synthesized secretion signal sequence, was linked with a ligase. This was cut again with NheI and XbaI enzymes, and the synthesized Mtb32A was linked with ligase. This was cut again with AscI and XbaI enzymes, and then the synthesized HA tag was linked with ligase to prepare pGX10 / tMtb32Amut.

Example 2: Construction of pGX10 / tFMtb32Amut DNA

Codon optimized Flt3L having the nucleic acid sequence of SEQ ID NO: 3 was chemically synthesized. Eco47III (5 ') and NotI-EcoRV (3') sites were added at the ends to facilitate insertion into the vector. PGX10 / tMtb32Amut prepared in Example 1 was treated with Eco47III and EcoRV enzyme to connect F with ligase to prepare pGX10 / tFMtb32Amut.

Example  3: pGX10 / tMtb32Amut  And pGX10 / tFMtb32Amut DNA Confirmation of expression of

To determine whether the Mtb32Amut, FMtb32Amut genes are expressed as proteins, transfecting the pGX10 / tMtb32Amut and pGX10 / tFMtb32Amut DNA vectors to COS7 cells in a usual manner and then Western blotting using HA tags according to conventional methods Was performed.

As a result, in the cells transduced with the pGX10 vector, which is an empty vector containing no Mtb32Amut gene, the Ag85A protein was not expressed, whereas the pGX10 / tMtb32Amut and pGX10 / tFMtb32Amut vectors into which the Mtb32Amut and FMtb32Amut genes were inserted were transduced. In the cell, a band of protein was identified in the size expected by HA tag.

Unlike the properties of Mtb32A known in the art through the present invention it can be confirmed that by mutating the active site of the serine protease can increase the expression rate in the cell. Therefore, the tuberculosis antigen showing a high preventive effect was built in a more readily available form, which can be usefully used for tuberculosis prevention because it can reduce the production cost and the risk of enzymatic toxicity (Fig. 1).

Example  4: pGX10 / tFMtb32Amut Immune response

In order to confirm the tuberculosis prevention effect by immunization of pGX10 / tFMtb32Amut, intramuscular injection (intra-muscular) of 5-6 weeks old female C57BL / 6J mice (SLC, Shijuoka, Japan) three times at four-week intervals was performed three times apart. Electroporation was applied after injection, and pGX10 (mock) and pGX10 / tFAg85A were intramuscularly injected at the same dose and interval as a control. Another control group was subcutaneous injection (BCG) once 2 weeks after the second intramuscular injection (FIG. 2).

In order to confirm the antigen specific immune response generated by pGX10 / tFMtb32Amut, FACS analysis via intracellular cytokine staining was performed. Twelve weeks after the first intramuscular injection, the rat lung and spleen were removed and 1 × 10 ⁶ cells were added with recombinant Ag85A protein, or Mtb32A CD8 T cell epitope, and placed on a 96 well round bottm plate. C was incubated in a 5% CO ₂ incubator. After 6 hours of initial culture, BFA (Brefeldin A, 10 / ml; Sigma, Deisenhofen, Germany) was added and cells were cultured for FACS analysis after 12 hours of further incubation at a concentration of 1 × 10 ⁷ cells / ml. (1% FCS, 0.02% sodium azide dissolved in PBS). First, the cells were stained with a cell surface recognition antibody, and then permeabilized buffer (dissolved with saponin at 0.5% in FACS buffer) to increase the surface permeability and stained with a cytokine recognition antibody. After staining was completed, cells were fixed with 1% formaldehyde. Flow cytometry was performed on FACScalibur BD (Biosciences, Mountain View, Calif.) Using the CellQuest software program. Specific T cell immune responses were measured by intracellular cytokine staining using an epitope or recombinant protein corresponding to each immunized antigen, indicating that pGX10 / tFMtb32Amut is a well-known tuberculosis antigen. Like pGX10 / tFAg85A, which expresses a peptide in the form of a fusion of Ag85A with its Flt3L, it induces a high antigen-specific immune response, which is characterized by a mutation of the enzyme and an improvement in Mtb32A through Flt3L fusion. It is shown to be effective in inducing. The first case of inducing expression and immune response with Mtb32A antigen alone is the first (FIG. 3) by the present invention.

Example  5: Mycobacterium tuberculosis preparation

The antigen for infection to be used in the latent infection model was prepared using Mycobacterium tuberculosis, Mycobacterium tuberculosis. Specifically, M tuberculosis H37Rv (ATCC 27294) was grown for 10 days with surface pellicles on Sauton medium, and the surface biofilms were collected and shaken with 3 mm glass beads and shaken slowly. The supernatant was collected after aggregation had settled, and the aliquot was stored at -70 ° C. After dissolving the stored aliquots, viable organisms were counted by serial dilution plating in Middlebrook 7H11 agar (Difco, Detroit, Mich.). To inoculate M. tuberculosis in mice, the suspension of the strain prepared above was briefly sonicated and diluted to 250,000 pfu / ml using PBS.

Example  6: pGX10 / tFMtb32Amut Tuberculosis prevention effect

Tuberculosis prevention effect by pGX10 / tFMtb32Amut was confirmed by aerosol infection of mice with Mycobacterium tuberculosis that causes tuberculosis after immunization.

The present inventors used two models to confirm tuberculosis prevention effect, one of which was a short-term prevention model to infect Mycobacterium tuberculosis 4 weeks after the last immunization, and the other was an infection of Mycobacterium tuberculosis 16 weeks after the last immunization model. Specifically, the immunized mice and control mice of the same age are placed in the inhalation chamber (Glas-Col, Terre Haute, Ind.) Of the airborne infection apparatus, followed by M. tuberculosis prepared in Example 5 above. 10 ml was exposed for 60 minutes at cis H37Rv 250,000 pfu / ml. When the above process is performed, human tuberculosis bacteria are naturally transmitted to the animal through air.

M. tuberculosis of non-immunized control mice was counted one day after exposure of Mycobacterium tuberculosis to determine if they were infected with Mycobacterium tuberculosis. Mice were euthanized with carbon dioxide and homogenized lungs and spleens in saline with 0.05% Tween 90 dissolved. Tissue suspensions were diluted 10-fold and 100 μl of each dilution was plated on M7H11 agar plates (Difco, Detroit, Mich.) And incubated at 37 ° C. for 3-4 weeks. After counting M. tuberculosis colonies on each plate, the results are expressed in log ₁₀ colony forming units (CFU). As a result, it was confirmed that about 200 M. tuberculosis were delivered to the lung.

The present inventors examined the tuberculosis prevention effect of pGX10 / tFMtb32Amut DNA vaccine through bacterial counting. Bacterial counts were determined from the tissues of mice after Mycobacterium tuberculosis infection at the time specified in each model.

As a result, the highest control bacteria remained in the lung and spleen of the mock (pGX10) without tuberculosis antigen, while the positive control group (BCG, pGX10-tF-Ag85A) showed lower bacterial residuals. The validity of the experimental conditions was provided. In particular, bacterial residuals due to BCG currently in use are accepted as baselines for conventional positive controls. Mice immunized with the experimental group (pGX10 / tFMtb32Amut) showed higher protective effects than pGX10-tAg85A, a positive control composed of the same single TB antigen in both lung and spleen, short-term and long-term models. In particular, compared to the current commercially available BCG, the long-term model shows a similar protective effect in the lung and spleen. Tuberculosis prophylaxis vaccines require long-term prophylaxis rather than short-term prophylaxis, so the present invention can be usefully used for the prevention of tuberculosis (FIGS. 4 and 5).

Abbreviations are defined as follows. "tPa" or "t" is secretory signal sequence of tissue plasminogen activator, "mut" is mutation of active site, "Co" is codon optimized nucleic acid sequence, "F" "Means Flt3L.

1 is an electrophoresis picture of Western blot analysis of the expression of FMtb32mut protein after transduction of pGX10-tFMtb32mut vector into COS7 cells.

Figure 2 is a schematic diagram showing the timing of DNA vaccine treatment and bacterial counts after aerosol infection in the short-term prevention model (A) and long-term prevention model (B) in the tuberculosis infection mouse model.

Figure 3 is a graph showing whether each antigen protein specific IFN-γ production by immune cells present in the lung (A) and spleen (B) of the mouse 4 weeks after the three DNA vaccinations.

Figure 4 is a graph showing the immunotherapy effect when the DNA vaccine was administered in the short-term prevention model of tuberculosis through bacterial counts (A: lungs of mice 4 weeks after aerosol infection, B: spleen of mice 4 weeks after aerosol infection) .

Figure 5 is a graph showing the immunotherapy effect when the DNA vaccine was administered in the long-term preventive model of tuberculosis through bacterial counts (A: lungs of mice 4 weeks after aerosol infection, B: spleen of mice 4 weeks after aerosol infection) .

<110> POSTECH FOUNDATION <120> A pharmaceutical composition for preventing tuberculosis <130> PA080056 / KR <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> codon optimized signal sequence of tissue plasminogen activator <400> 1 ggtaccatgg acgccatgaa gcgcggcctg tgctgcgtgc tgctgctgtg cggcgccgtg 60 tttgtgagcc ccagcgctag c 81 <210> 2 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> codon optimized mycobacterium tuberculosis Mtb32Amut <400> 2 gctagcgata tcatggcccc tcccgccctg agccaggaca gatttgccga ctttcctgcc 60 ctgcccctgg accctagcgc cttggtggcc caggtgggac cccaggtggt gaacatcaac 120 accaagctgg gctacaacaa cgccgtgggc gctggcacag gcatcgtgat cgaccccaat 180 ggcgtggtgc tgaccaacaa cgccgtgatc gccggagcca cagacatcaa cgcctttagc 240 gtgggcagcg gccagaccta cggcgtggac gtggtgggct acgatagaac ccagaacgtg 300 gccgtgctgc agctgagagg cgctggcgga ctgcccagcg ccgccatcgg aggcggcgtg 360 gccgtgggcg agcccgtggt ggccttgggc aacagcggag gccagggcgg aacccctaga 420 gccgtgcctg gcagagtggt ggccctgggc cagaccgtgc aggccagcga cagcctgaca 480 ggcgccgagg agaccctgaa tggcctgatc cagtttgacg ctgccatcca gcctggcgat 540 gccggcggac ccgtggtgaa tggcctgggc caggtggtgg gcatgaacac agccgccagc 600 gacaactttc agctgagcca gggcggccag ggctttgcca tccccatcgg ccaggccttg 660 gccatcgccg gccagatcag aagcggcgga ggcagcccca ccgtgcacat cggccccaca 720 gcctttctgg gcctgggcgt ggtggacaac aacggcaacg gcgctagagt gcagagagtg 780 gtgggcagcg cccctgctgc cagcctgggc atcagcaccg gcgacgtgat cacagccgtg 840 gacggagccc ccatcaacag cgctaccgcc ttggccgatg ccctgaacgg ccatcaccct 900 ggcgacgtga tcagcgtgac ctggcagacc aagagcggcg gaacaagaac cggcaacgtg 960 accctggccg agggccctcc cgctactagt ggcgcgccat ctaga 1005 <210> 3 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> codon optimized Flt3L <400> 3 agcgctatga ccgtgctggc ccccgcctgg agccccaaca gcagcctgct gctgctgctg 60 ctgctgctga gcccctgcct gaggggcacc cccgactgct acttcagcca tagccccatc 120 agcagcaact tcaaggtgaa gttcagggag ctgaccgacc atctgctgaa ggactacccc 180 gtgaccgtgg ccgtgaacct gcaggacgag aagcattgca aggccctgtg gagcctgttc 240 ctggcccaga ggtggatcga gcagctgaag accgtggccg gcagcaagat gcagaccctg 300 ctggaggacg tgaacaccga gatccatttc gtgaccagct gcaccttcca gcccctgccc 360 gagtgcctga ggttcgtgca gaccaacatc agccatctgc tgaaggacac ctgcacccag 420 ctgctggccc tgaagccctg catcggcaag gcctgccaga acttcagcag gtgcctggag 480 gtgcagtgcc agcccgacag cgcggccgca gatatc 516 <210> 4 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> HAtag <400> 4 ggcgcgccct acccatacga tgttccagat tacgcttacc cctacgacgt gcccgactac 60 gcctaagcgg ccgccacgat cgtggtctag a 91

Claims (13)

(1) a nucleic acid sequence of SEQ ID NO: 1 encoding a secretion signal sequence and (2) a nucleic acid sequence of SEQ ID NO: 3 encoding a Flt3L (fms-like tyrosine kinase 3 ligand) linked to the 3 'end of the secretion signal sequence, and (3) a nucleic acid having the nucleic acid sequence of SEQ ID NO: 2 encoding the active site mutant of Mtb32A of Mycobacterium tuberculosis linked to the 3 'end of Flt3L. delete delete delete delete delete delete delete delete A pharmaceutical composition for preventing tuberculosis comprising the nucleic acid of claim 1. The pharmaceutical composition of claim 10, wherein the nucleic acid is provided in the form of an expression vector. The pharmaceutical composition of claim 11, wherein the expression vector is an expression vector derived from a virus, plasmid, bacteriophage, or cosmid. The pharmaceutical composition of claim 10, wherein the composition is in the form of a vaccine.

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